Process for encapsulating an active lipid-soluble substance by preparing a pit emulsion and emulsion obtained

ABSTRACT

The invention relates to a method of encapsulating an active lipid-soluble substance in nanocapsules, by preparing an emulsion. The inventive method is characterised in that it consists in: (a) obtaining an aqueous phase and an oil phase; (b) raising the temperature of the two phases to a temperature greater that the phase inversion temperature; (c) mixing the two phases; (d) incorporating the active lipid-soluble substance into the lipid-soluble phase; (e) allowing the temperature to decrease to the phase inversion temperatures; (f) once the phase inversion is effective and the emulsion is in a continuous aqueous phase, quenching the emulsion obtained in order to lower the temperature thereof. The invention also relates to the emulsion that can be obtained using the inventive method, said emulsion being characterised in that the average nanocapsule size is less than 300 nm.

The present invention relates to the field of vectorizing activeprinciples.

The efficacy of a formulation both in pharmacy and in cosmetics dependson the active principles but also on their release system, and manyvectorization means have been explored either in cosmetics or inpharmacy.

Among these, mention may be made of nanoparticles. Nanoparticles arecolloidal particles ranging from 1 to 1000 nm in size. They aremacromolecules in which the active principle is dissolved, trapped orencapsulated. These nanoparticles refer to very different systems, forinstance nanospheres and nanocapsules, which are, respectively, matrixsystems for the nanospheres, and reservoir systems for the nanocapsules.

Nanospheres are solid matrix particles in which the active principle isfinely dispersed in the polymer matrix.

Nanocapsules are particles consisting of a core that is liquid orsemiliquid at room temperature, which contains the active principle,coated with a film that is solid at room temperature.

The present invention more particularly relates to the field ofvectorizing liposoluble active principles in a reservoir system ofnanocapsule type. Nanocapsules are aqueous suspensions of small vesicles(generally between 100 and 400 nm), the thin rigid wall of whichconsists of macromolecules of natural, synthetic or semisyntheticorigin. These systems allow the encapsulation in the lipophilic core ofrelatively large amounts of active principles, which are usuallylipophilic, and may be obtained either via polymerization reactions orfrom preformed polymers. Many processes for formulating nanocapsules byemulsification are described, and examples that will be mentionedinclude the processes described in patents U.S. Pat. No. 5,079,322 or EP0 717 989, for obtaining emulsions incorporating liposoluble activeprinciples. The term “liposoluble active principles” in particular meansany chemical compound or mixture that is soluble in oily substances usedin cosmetics, the food sector, pharmaceuticals or the veterinary sectoror any compound that is advantageous as a result of its properties. Someliposoluble active principles are sensitive to exposure to temperaturesabove 50° C., and sensitive to light and to oxidation. One of thesolutions currently used for vectorizing these active principles is toformulate them in emulsions. However, on account of their instability,when these liposoluble active principles are used in emulsified systems,they are introduced at the end of the process into an oil-in-wateremulsified system at a temperature below 50° C., for example, and theythen become randomly distributed, particularly in the aqueous phase andwill then be partially destroyed by the surrounding medium.

These processes are therefore not entirely satisfactory, either becausethe amounts of active principles incorporated are insufficient toachieve the desired activities, or because the stability is not correct,or even because the production processes are difficult to implementindustrially.

To improve these formulations, processes of emulsification by phaseinversion, known as “PIT (Phase Inversion Temperature) emulsion”, forinstance those described in patents WO 20011975, EP 1 093 795 or WO200071676 for obtaining oil-in-water emulsions containing an activeprinciple, have been proposed.

These processes include the incorporation, for example, of an activeprinciple into an oily phase, the addition of some of the aqueous phaseto the mixture obtained, heating with stirring to a temperature abovethe phase inversion temperature, addition of the remainder of theaqueous phase, and cooling. For example, WO 200164328 discloses aprocess for preparing lipid nanocapsules based on the phase inversion ofan oil/water emulsion induced by several cycles of raising and reducingthe temperature. The emulsions obtained are very fine and do not requirehomogenization steps. These processes allow the production of very finedispersions of the emulsion (0.1 to 0.3 μm) and great stability, since,during the phase inversion, the interface tension is minimal and allowsvery fine droplets to be obtained. However, the phases of temperatureincrease to obtain the phase inversion, which may optionally berepeated, are incompatible with the formulation of active principlesliable to undergo physical or chemical degradation due to excessiveexposure to a temperature above 50° C.

In the present invention, the lipid nanocapsules are formulated via aprocess of emulsification by phase inversion induced by passing theemulsion above the phase inversion temperature, but which allows theactive principle to be preserved by incorporating it into the oilycontinuous phase, and thus without contact with the aqueous phase, abovethe phase inversion temperature.

Specifically, the incorporation of the liposoluble active principle intothe formulation, at a temperature above the phase inversion temperature,i.e. when the emulsion is in the oily continuous phase (water-in-oilemulsion), makes it possible to obtain a good distribution of the activeprinciple in the oily phase, limits its contact with the aqueous phase,and, surprisingly, although the temperature is high, since the residencetime at this temperature is very short because this incorporation isfollowed by annealing of the emulsion, the degradation phenomena arelimited or eliminated.

The present invention relates to a process for encapsulating aliposoluble active principle in nanocapsules by preparing an emulsion,characterized in that:

-   a) an aqueous phase and a fatty phase are provided,-   b) the temperature of the two phases is raised to a temperature    above the phase inversion temperature,-   c) the two phases are mixed together,-   d) the liposoluble active principle is incorporated into the    liposoluble phase,-   e) the temperature is lowered to the phase inversion temperature,-   f) once the phase inversion is effective and the emulsion is in the    aqueous continuous phase, the emulsion obtained is annealed to lower    its temperature.

In one variant after step c), a step c′) is performed, which consists inlowering the temperature to a temperature immediately above the phaseinversion temperature before incorporating the active principle.

This lowering of temperature may be induced or may take place naturally.

In one variant, the temperature may be left to lower naturally or thetemperature may be lowered to a desired temperature by performing anannealing operation.

The invention also relates to a process according to the claim,characterized in that step c) is performed before step b). In thisprocess variant, the mixing of the two phases is performed beforeraising the temperature or during the raising of temperature, but beforethe temperature reaches the phase inversion temperature. The emulsionobtained is then brought to a temperature above the phase inversiontemperature, and the active principle is then incorporated.

In one variant of the process according to the invention, the emulsionobtained is then concentrated by withdrawal of some of the aqueousphase.

Advantageously, this concentration step may be performed by tangentialultrafiltration.

According to the invention, the “annealing” step f) is performed byadding an additional amount of aqueous phase brought to a temperature atleast below the phase inversion temperature, and optionally below roomtemperature. This sudden and rapid cooling step makes it possible tolower the temperature of the emulsion and to reduce the time of exposureof the active principle to a raised higher temperature.

This annealing may also be performed using a heat-exchange coolingsystem or by adding liquefied gas, for example nitrogen.

The term “temperature immediately higher than the phase inversiontemperature” means a temperature a few degrees higher, in practice 1 or2° C. higher than the phase inversion temperature, the phase inversiontemperature of the system having been determined experimentallybeforehand by monitoring the conductivity of the system or by visualobservation.

Among the active principles that may be encapsulated via this process,mention will be made more particularly of “unstable” liposoluble activeprinciples, i.e. active principles liable to degrade if they are exposedto temperatures above 40° C. for longer than 30 minutes, or activeprinciples that are sensitive to oxidation due to the presence of waterin the formulation, or that are degraded by pH variations, UV radiationor the presence of products liable to cause side reactions with saidactive principles.

Among the liposoluble active principles that may be encapsulated viathis process, examples that will be mentioned include:

-   -   liposoluble vitamins and derivatives thereof, such as the        retinoid family (retinol, retinaldehyde and retinoic acid), the        carotenoid family, and tocopherol and its derivatives,    -   polyphenols such as flavonoids (e.g.: isoflavonoids, quercetin),        stilbenes (e.g.: resveratrol), catechins (e.g.: epicatechin        3-galate, epigallocatechin 3-gallate),    -   fragrance components, for instance vanillin, indole, and more        generally essential oils such as essential oils of citrus fruit        or of lavender,    -   liposoluble pharmaceutical active principles such as:        fluvastatin, ketoprofen, verapamil, atenolol, griseofulvin,        ranitidine.

In the process according to the invention, the emulsion comprises from5% to 30% of fatty substance constituting the fatty phase and from 45%to 92% of water constituting the aqueous phase. The proportion of thefatty phase relative to the aqueous phase associated therewith dependson the amount of active principle to be encapsulated and on the type ofemulsion. The proportion of fatty phase may also have an influence onthe size of the nanocapsules obtained.

The constituents of the fatty phase may be chosen from paraffinderivatives or more or less complex triglycerides. The choice of theseconstituents will depend on the nature of the lipophilic activeprinciple to be encapsulated, but also on their potential influence onthe phase inversion temperature, or even on their influence on the sizeof the nanocapsules obtained.

The nature of the active principle to be encapsulated will have aninfluence on the choice of constituents of the fatty phase, since theconstituents will be selected as a function of:

-   -   the potential solubility of the active principle in this phase,    -   their neutrality with respect to the active principle, i.e. they        must not be oxidizing with respect to the active principle, i.e.        they must have a low acid number, must not be acidic and must        have a low iodine number,    -   their compatibility with a phase inversion emulsification        technique,    -   their ability to give the lowest possible phase inversion        temperature.

When the phase inversion temperature is too high, ingredients capable oflowering this phase inversion temperature will be added to the medium.

Specifically, the more pronounced lipophilic nature of certainconstituents liable to be chosen on account, for example, of theirability to dissolve the active principles may lead to an increase in thephase inversion temperature, since the enhancement of the hydrophobicbonds between the surfactant and the oil leads to an increase in theenergy required to invert the system. The polarity of the constituentsof the fatty phase also has an influence on the phase inversiontemperature: the more polar the constituents, the higher the phaseinversion temperature. On the other hand, saturated constituents, withthe lowest possible iodine number, are capable of reducing the phaseinversion temperature.

Although the residence time at a temperature above the phase inversiontemperature is extremely short, it will nevertheless be sought toformulate emulsions whose phase inversion temperature is as low aspossible.

The constituents of the fatty phase will thus preferably be chosen frommineral oils or mineral oil substitutes such as isohexadecane,silicones, especially cyclomethicones or polydimethylsiloxane, C8 to C12triglycerides, for example capric and caprylic acid triglycerides, andmixtures thereof.

The choice of the emulsifying system is also an important criterion thathas an influence on the stability of the emulsions obtained and on theparticle size. Two values characterize an emulsifying system, thelipophilic surfactant/hydrophilic surfactant ratio (LS/HS ratio) and theoverall percentage of surfactants.

The emulsifying systems used in the present invention will be chosenfrom systems whose LS/HS ratio is between 1/1 and 1/50. The percentageof water-soluble surfactant will preferably be between 2% and 10% andthe percentage of lipophilic surfactant will preferably be between 1%and 5%.

The water-soluble surfactants are especially chosen from glycol esters,glycerol esters, itol esters, sorbitan esters and polyethylene glycolesters. Among the polyethylene glycol esters that will especially bechosen are those whose carbon-based chain is between 10 and 22 carbonatoms and for which the number of polyethylene glycol monomers isbetween 5 and 30. These water-soluble surfactants may also be chosenfrom fatty alkyl ethers of polyethylene glycol, whose fatty alcohol ischosen from those containing from 10 to 22 carbon atoms and whosemonomer number is between 5 and 30.

Lipophilic surfactants will also be added to the mixture; thesesurfactants are characterized by their ability to give W/O emulsionswhen used as emulsifiers alone or predominantly. Among theseemulsifiers, mention will be made of monoglycerol esters andpolyglycerol esters of fatty acids, silicone emulsifiers such as cetyldimethicone copolyol, and polyhydroxystearic acid esters of polyethyleneglycol.

According to one embodiment of the invention, the salt may be added tothe aqueous phase. It has been demonstrated that the addition of saltreduces the interaction between the polar groups and the water andreduces the hydrophilicity of the surfactant, and thus the CMC. Inaddition, it produces a screen effect that facilitates approach betweenthe polar groups.

Moreover, studies have revealed that modification of the saltconcentration results in a displacement of the phase inversion zone. Thehigher the salt concentration, the lower the phase inversiontemperature.

Other constituents may be added to one or other of the phases; examplesthat will be mentioned include preserving agents for preventing thegrowth of certain microorganisms in the aqueous phase.

The antioxidants are added to the system to prevent impairment ofcertain readily oxidizable compounds in the lipid phase. They arechosen, for example, from the group consisting of butylhydroxyanisole(BHA), butylhydroxytoluene (BHT), propyl gallate, α-tocopherol and EDTA.These antioxidants will be used in concentrations ranging from 0.01% to3%; for example, BHT will be used in concentrations ranging from 0.01%to 1%, α-tocopherol in concentrations ranging from 0.1% to 3% and EDTAin concentrations ranging from 0.05% to 2%.

In the process according to the invention, the stirring speed will bebetween 100 and 3000 rpm. Specifically, during the emulsification, adynamic equilibrium is established between rupture (zones at high shear)and coalescence (zones at low shear). The stirring speed affects therupture and the coalescence, and this stirring speed will thus have aninfluence on the size distribution and the stability of the emulsion.

In the process according to the invention, detection of the phaseinversion is performed:

-   -   either by visualization of the formulation: the organization of        the system in the form of nanoparticles is reflected visually by        a change in the appearance of the initial system, which goes        from opaque-white to translucent-white. For poorly dispersed        emulsions, the appearance occasionally becomes bluish during the        phase inversion,    -   or by measuring the conductivity, which increases when the        emulsion passes from a water-in-oil system to an oil-in-water        system.

Specifically, the conductivity increases when the emulsion passes from awater-in-oil system to an oil-in-water system. An electrolyte-richaqueous continuous phase is characterized by a high conductivity value.The PIT zone is defined as being a zone in which the conductivity of themedium changes from a zero value (characterizing an oily continuousphase) to a value of a few μs/cm. This change takes place over atemperature range known as the PIT zone.

The particle diameter is measured via an optical method of lightmeasurement known as light scattering, which is based on variousphysical and mathematical laws including PCS (Photon CorrelationSpectroscopy). The principle of the measurement may be described as astudy of the speed of particles subjected to Brownien motion, the smallparticles vibrating considerably and moving quickly, whereas those oflarger diameter vibrate little and move more slowly. The interaction ofa light beam with the particles makes it possible, after mathematicalmodeling, to estimate the particle diameter.

The present invention also relates to lipid nanocapsules obtained viathe process according to the invention, the mean size of which is lessthan 300 nm and preferably on average 150 nm.

Emulsions according to the invention are described below.

EXAMPLE 1

A fatty phase containing the following ingredients is formulated:tocopheryl acetate (vitamin E acetate) 0.5%   glyceryl stearate andceteareth-12 and ceteareth-20 and cetearyl 3% alcohol (Emulgade SEV)ceteareth-20 (Eumulgin B2) 2% isohexadecane (Arlamol HD) 6%cyclomethicone (Dow Corning 345) 3% butylhydroxytoluene (BHT) 0.1%  

An aqueous phase containing the following ingredients is formulated:sodium salt of EDTA (BASF (disodium EDTA)) 0.5% demineralized water  25%

The two phases formulated above are heated to a temperature of 85° C.

The two phases are combined by adding the aqueous phase to the fattyphase with shearing stirring at 700 rpm.

The active principle retinol, as a 7% solution in a caprylic acidtriglyceride, is then incorporated into the emulsion obtained by mixingtogether the aqueous phase and the fatty phase at a temperature in theregion of 81° C.

The phase inversion takes place at 73° C., this phase inversion beingdetected by an increase in the conductivity of greater than 1 μS/cm.

An additional aqueous phase containing a preserving agent, Glydant PlusLiquid (DMDM hydantoin and iodopropynyl butylcarbamate (sold by thecompany Lonza Inc. (0.5%) and water 51.9% is rapidly incorporated intothe emulsion obtained above containing the retinol.

The emulsion may then be concentrated by tangential ultrafiltration.

EXAMPLE 2

According to the same procedure as in example 1, an emulsion is preparedstarting with the following phases:

Fatty Phase: PEG-30 dipolyhydroxystearate 2% PEG-6 stearate andceteth-20 and steareth-20 6% isohexadecane 6% cyclomethicone 3%tocopheryl acetate 0.5%   butylhydroxytoluene 0.1%  

Aqueous Phase: disodium EDTA 0.2% demineralized water  25%Active Principle:retinol, as a 7% solution in a caprylic acid triglyceride

The phase inversion takes place at 71° C.

Additional Aqueous Phase: chlorhexidine digluconate 0.5% water 49.7% 

EXAMPLE 3

According to the same procedure as in example 1, an emulsion is preparedfrom the following phases:

Fatty Phase: PEG-30 dipolyhydroxystearate 2% PEG-6 stearate andceteth-20 and steareth-20 6% isohexadecane 6% cyclomethicone 3%tocopheryl acetate 0.5%   butylhydroxytoluene 0.1%   caprylic/caprictriglyceride 6%

Aqueous Phase: disodium EDTA 0.2% demineralized water  25%Active Principle:retinol, as a 0.33% solution in a caprylic acid triglyceride

The phase inversion takes place at 80° C.

Additional Aqueous Phase: chlorhexidine digluconate 0.5% sodiummethylparaben 0.2% water 50.17% 

Among the advantages of the process according to the invention, mentionmay be made of the size of the droplets obtained, of less than 300 nm,which has the following advantages:

-   -   improved bioavailability of the incorporated active principle,        since the penetration of the emulsion is promoted by the minimal        size of the particles encapsulating the active principle, this        improved bioavailability of the incorporated active principle        allows the final concentration in the product to be lower than        with standard encapsulating systems and reduces the possible        side effects,    -   better physical stability of the finished product; specifically,        the smaller the particle size, the more physically stable the        system on account of the disappearance of the maturation and        coalescence phenomena,    -   the production of monodisperse systems (polydispersity        index<0.25): since the size of nanocapsules is homogeneous, the        Oswald maturation is limited,    -   manufacturing processes that are faster and more economical than        the standard emulsification processes on account of the        reduction in the energy required.

1-8. (canceled)
 9. A process for encapsulating a liposoluble activeprinciple in nanocapsules by preparing an emulsion, characterized inthat: a) an aqueous phase and a fatty phase are provided, b) thetemperature of the two phases is raised to a temperature above the phaseinversion temperature, c) the two phases are mixed together, d) theliposoluble active principle is incorporated into the liposoluble phase,e) the temperature is lowered to the phase inversion temperature, f)once the phase inversion is effective and the emulsion is in the aqueouscontinuous phase, the emulsion obtained is annealed to lower itstemperature.
 10. The process as claimed in claim 9, characterized inthat step c′) is performed, which consists in lowering the temperatureto a temperature immediately above the phase inversion temperaturebefore incorporating the active principle.
 11. The process as claimed inclaim 9, characterized in that step c) is performed before step b). 12.The process as claimed in claim 9, characterized in that the emulsionobtained is then concentrated by withdrawal of some of the aqueousphase.
 13. The process as claimed in claim 9, characterized in that stepe) is performed by adding an additional amount of aqueous phase broughtto a temperature below the phase inversion temperature.
 14. The processas claimed in claim 9, characterized in that the active principle isdissolved in an additional amount of fatty phase before beingincorporated into the system.
 15. The process as claimed in claim 9,characterized in that the active principle is chosen from the groupconsisting of liposoluble vitamins such as retinol, retinoids, vitamin Eand carotenoids, polyphenols and fragrance components.
 16. An emulsionthat may be obtained via a process as claimed in claim 9, characterizedin that the size of the nanocapsules is on average less than 300 nm.